EFFECTS OF ARABINOSYLCYTOSINE-SUBSTITUTED DNA ON DNA RNA HYBRID STABILITY AND TRANSCRIPTION BY T7 RNA-POLYMERASE/

Cytosine arabinoside (araC) is a potent antileukemic agent which inter feres with DNA replication both as a dNTP competitive inhibitor as wel l as after its misincorporation into DNA. We previously developed a ch emical methodology for the synthesis of DNA oligomers containing araC which allowed us to study its site specific effects on duplex stabilit y and chemical reactivity [Beardsley, G. P., Mikita, T., Klaus, M., an d Nussbaum, A. (1988) Nucleic Acids Res. 16, 9165], as well as its eff ects on DNA ligase and DNA polymerase activity [Mikita, T., and Beards ley, G. P. (1988) Biochemistry 27, 4698]. The DNA polymerase studies, in addition to other observations, showed that araC in DNA templates c ould have an inhibitory effect on polymerase bypass. As a template les ion, there exists the potential for interference with other aspects of DNA metabolism, such as transcription. We have characterized a DNA/RN A hybrid containing an araC-G base pair, comparing thermal stability, chemical cleavage rates, and duplex gel mobility to an identically seq uenced DNA duplex. We find that the A-form DNA/RNA hybrid and the B-fo rm DNA duplex are nearly identical in the extent their thermal stabili ty is affected by an araC-G(dG) base pair. Substitutions of araC for d C were made at various positions in a series of DNA duplex substrates containing a T7 RNA polymerase promoter with variable length coding st rands. These were used to probe the effect of araC on promoter recogni tion, initiation, and elongation by T7 RNA polymerase in vitro. Substi tutions in the central promoter region had no observable effect on RNA polymerase binding, initiation rate, or transcriptional output. Codin g strand substitutions defined an area of high sensitivity in the init iation region where miss-starts, primer slippage, and an inability to escape from abortive cycling occur depending on the position substitut ed. Substitutions after position 10 had little effect on transcription output. These highly variable, position dependent effects indicate a narrow window of vulnerability where transcription output is severely reduced (similar to 100-fold) by a subtle DNA lesion that has little o r no consequence when situated elsewhere in these small coding units.